Mutation spectrum of hearing loss patients in Northwest China: Identification of 20 novel variants

Abstract Background Hearing loss (HL) is the most frequent sensory deficit in humans, with strong genetic heterogeneity. The genetic diagnosis of HL is very important to aid treatment decisions and to provide prognostic information and genetic counselling for the patient's family. Methods We detected and analysed 362 Chinese non‐syndromic HL patients by screening of variants in 15 hot spot mutations. Subsequently, 40 patients underwent further whole‐exome sequencing (WES) to determine genetic aetiology. The candidate variants were verified using Sanger sequencing. Twenty‐three carrier couples with pathogenic variants or likely pathogenic variants chose to proceed with prenatal diagnosis using Sanger sequencing. Results Among the 362 HL patients, 102 were assigned a molecular diagnosis with 52 different variants in 22 deafness genes. A total of 41 (11.33%) cases with the biallelic GJB2 (OMIM # 220290) gene mutations were detected, and 21 (5.80%) had biallelic SLC26A4 (OMIM # 605646) mutations. Mitochondrial gene (OMIM # 561000) mutations were detected in seven (1.93%) patients. Twenty of the variants in 15 deafness genes were novel. SOX10 (OMIM # 602229), MYO15A (OMIM # 602666) and WFS1 (OMIM # 606201) were each detected in two patients. Meanwhile, OSBPL2 (OMIM # 606731), RRM2B (OMIM # 604712), OTOG (OMIM # 604487), STRC (OMIM # 606440), PCDH15 (OMIM # 605514), LOXHD1 (OMIM # 613072), CDH23 (OMIM # 605516), TMC1 (OMIM # 606706), CHD7 (OMIM # 608892), DIAPH3 (OMIM # 614567), TBC1D24 (OMIM # 613577), TIMM8A (OMIM # 300356), PTPRQ (OMIM # 603317), SALL1 (OMIM # 602218), and GSDME (OMIM # 608798) were each detected in one patient. In addition, as regards one couple with a heterozygous variant of CDH23 and PCDH15, respectively, prenatal diagnosis results suggest that the foetus had double heterozygous (DH) variants of CDH23 and PCDH15, which has a high risk to cause ID/F type Usher syndrome. Conclusion Our study expanded the spectrum of deafness gene variation, which will contribute to the genetic diagnosis, prenatal diagnosis and the procreation guidance of deaf couple. In addition, the deafness caused by two genes should be paid attention to in the prenatal diagnosis of families with both deaf patients.


| BACKGROUND
Hearing loss (HL) is one of the most prevalent sensory deficits in humans, with a prevalence of about 2.5/1000 in birth (Abou Tayoun et al., 2016).More than half of HL individuals are affected by genetic factors (Nance et al., 2006).HL includes non-syndromic HL (NSHL), where the hearing impairment is the only clinical feature, and syndromic with other abnormalities (Zhou et al., 2019).Genetic diagnosis of NSHL is important for the diagnosis, intervention and prevention of inherited HL in patients' families (Yang et al., 2019).The genetic mode of NSHL is usually subdivided into autosomal recessive (approximately 77%), autosomal dominant (approximately 22%), mitochondrial (approximately 1%) and X/Y-linked (less) (ACMG, 2002).To date, at least 224 genes have been reported to be associated with HL in humans (https:// morl.lab.uiowa.edu/ genes -inclu dedotosc ope-v9), 66 are autosomal dominant, 117 are autosomal recessive, 21 are autosomal dominant/autosomal recessive, 9 are mitochondrial and 5 are X-linked (Jin et al., 2022).
The rapid development of next-generation sequencing (NGS) makes it possible to analyse all genes in one test.Whole-exome sequencing (WES), a platform of NGS, offers powerful applications for diagnosis as well as for identifying rare variants or new causative genes (Liang et al., 2021).
Here, we recruited 362 Chinese HL families with nonsyndromic hearing loss.Subsequently, we performed DNA microarray test targeting 15 deafness mutations and WES to investigate the contributing genetic factors in patients, which extended the spectrum of deafness-associated gene variants and provided further guidance for its positive intervention and cure.We provided invasive prenatal diagnosis for 23 carrier couples with pathogenic variants or likely pathogenic variants and helped them deliver healthy babies.

| Recruitment of patients
Three hundred and sixty-two patients with nonsyndromic HL were recruited from the outpatient clinic of the Department of Medical Genetics, Gansu Maternity and Child Health Care Hospital (Lanzhou, Gansu, China) in the period 2018-2023.These patients were clinically diagnosed with bilateral sensorineural hearing loss, and audiological tests were performed in the Department of Otolaryngology of the Gansu Maternity and Child Health Care Hospital.Tests included puretone audiometry (PTA, or behavioural audiometry) for patients >4 years old and multiple-frequency auditory steady-state evoked response (ASSR) tests for patients ≤4 years old (Guo et al., 2020).All probands were from unrelated Chinese families and some of them were couples trying to have children.The 362 Chinese HL patients included 192 males and 170 females, and they were aged from 3 days to 53 years, and the age of onset ranged from birth to 22 years.This study was undertaken according to the tenets of the Declaration of Helsinki 1975 and its later amendments.The study protocol was approved by the Ethics Committee of the Gansu Provincial Maternity and Child-Care Hospital (2021GSFY [65]).Written informed consent was obtained from all study participants or their legal guardians.

| Genomic DNA extraction
Peripheral blood samples (2-3 mL) were collected from the 362 probands and the parents of 40 patients.Genomic DNA was extracted using a Tiangen DNA extraction kit (Tiangen Biotech, Beijing, China), according to the manufacturer's instructions.DNA purity and concentration were determined by NanoDrop 2000 Spectrophotometer (Thermo Scientifc, USA).The concentration of the qualified sample was 50-250 ng/μL and the absorbance (OD260/OD280) read was 1.8-2.0.

Sanger sequencing
WES was performed by Chigene Co., Ltd. using the GenCap™ Custom Enrichment kit (MyGenostics, Beijing, China).The qualified genomic DNA was randomly fragmented to an average size of 180-280 bp by enzyme digestion.Next, the DNA fragments were end-repaired and phosphorylated, followed by A-tailing and ligation at the 3′-ends with paired end adaptors and index (Illumina).The size distribution and concentration of the libraries were determined by Qubit 3.0 Fluorometer and Agilent 2100 Bioanalyzer system.The library DNA was hybridized with the biotin-specific probe, and the magnetic beads, which were modified by streptavidin, captured the target gene.Finally, the DNA library was sequenced on NovaSeq 6000 (Illumina, CA, USA).Candidate variants were validated in the proband's parents in each family by Sanger sequencing.Sequencing primers were designed using online Primer Designer Tool Primer 3.0 (http:// prime r3.ut.ee) and genomic DNA was amplifed using the HotStarTaq Master Mix Kit.The PCR products were bidirectionally sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3500 Dx Genetic Analyzer (Applied Biosystems, USA).Data analysis was conducted using the software SeqMan Pro.
Among the remaining 293 HL probands, we recruited 40 HL families for WES to confirm the diagnosis.
T A B L E 1 Variants of screening in 15 hot spot mutation among 362 probands with hearing loss.
We identified 20 novel variants in 15 deafness genes, which were not previously reported in ClinVar or HGMD.According to the ACMG guidelines, 12 variants were classified as pathogenic variant, 4 were likely pathogenic variant and other 4 variants were of uncertain significance (Table 3).

| Prenatal diagnosis
After receiving the genetic counselling, 23 carrier couples with pathogenic variants or likely pathogenic variants chose to proceed with prenatal diagnosis.As shown in Table 4, seven foetuses harboured neither of the deafnesscausing variants from their parents and 9 foetuses were monoallelic variant carriers.Seven foetuses carried diallelic or compound heterozygous variants associated with autosomal recessive hearing loss, and their parents were provided with detailed information and early intervention programmes.

| DISCUSSION
In this study, we performed a molecular diagnosis of 362 unrelated Chinese with HL by screening of variants in 15 hot spot mutations and WES.We determined 52 different variants in 22 deafness genes.We identified 20 novel variants in 15 deafness genes, which were not previously reported in ClinVar or HGMD.
Liu et al. analysed the variant screening results of GJB2, GJB3, SLC26A4 and MT-RNR1 in 398 symmetrical sensorineural HL patients.Sixty-nine (17.34%) cases had the biallelic GJB2 gene variants, and the most common variants were c.235delC, c.109G > A and c.299_300delAT, with allele frequencies of 12.31%, 3.38% and 3.89%, respectively.Sixty-three (15.83%) cases with biallelic SLC26A4 variants were detected, and the most common pathogenic alleles were c.919-2A > G, c.2168A > G and c.1174A > T, with allele frequencies of 9.17%, 2.26% and 0.88%, respectively.Mitochondrial gene variants were detected in nine (2.26%) patients, with five cases of mitochondrial DNA (mtDNA) m.1555A > G variant and four cases of mtDNA m.1095 T > C variant (Liu et al., 2020).Hu et al. recruited 3541 subjects and used multiplex polymerase chain reaction (PCR) combined with next-generation sequencing to detect 100 hot spot variants in 18 common deafnessrelated genes.Thirty-seven alleles of eight deafness genes were detected.One hundred forty-five (4.09%) were found T A L E 2 Variant analysis of the HL patients with WES in this study.to be GJB2 gene variant carriers, and the hot spot variant was c.235delC (1.54%).Twenty-three (0.65%) were found to be GJB3 gene variant carriers.One hundred thirtytwo (3.37%) were found to be SLC26A4 gene variant carriers, and the hot spot variant was c.919-2A > G (0.49%).Forty-four (1.24%) were found to be mitochondrial DNA (mtDNA) variant carriers (Hu et al., 2021).

Age of diagnoses Gene
In our study, GJB2 mutations were detected in 46 of the 362 probands (12.7%), and the most common variants were c.235delC, with allele frequencies of 8.01%.In addition, 29 probands of SLC26A4 gene mutations were detected in 362 probands, accounting for 8.01%, and the most common pathogenic alleles were c.919-2A > G, with allele frequencies of 4.56%.Particularly, we identified a novel variant c.410C > T in SLC26A4 gene, which was not reported in ClinVar or HGMD previously.According to the ACMG guidelines, this variant was categorized as "likely pathogenic" variant.Furthermore, the mitochondrial gene mutations were found in seven patients, accounting for 1.93%, and GJB3 was detected in one patient (0.28%).Nineteen patients (5.25%) had other known deafness gene variants.The common variant genes and loci detected in this study were different from those detected in other regions or ethnic groups, which suggested that genetic screening or testing programmes for deafness should be formulated in accordance with the genetic characteristics of the region (Ma et al., 2023).
Cochlear implantation (CI) is an effective form of hearing restoration that improves quality of life and ameliorates the associated economic, social, emotional and neurocognitive consequences of severe-toprofound hearing loss.Some patients, however, do not obtain the expected benefit from implantation (Seligman et al., 2022).Variants in deafness genes lead to different pathologies and might result in varied CI outcomes.GJB2 and SLC26A4 gene variants are highly prevalent in prelingual HL patients from China.Patients with GJB2 or SLC26A4 variants showed better post-implant auditory performance and speech intelligibility than those without the variants only when implanted before the age of 3.5 years (Wu, Ko, et al., 2015).It has been demonstrated that variants in the TMPRSS3 (OMIM # 605511) gene were associated with poor CI outcome (Eppsteiner et al., 2012), whereas variants in the MYO15A, TECTA (OMIM # 602574), and ACTG1 (OMIM # 102560) genes also showed relatively good auditory performance after implantation (Miyagawa et al., 2013).Moreover, variants in the PCDH15 and DFNB59 (OMIM # 610219) genes were associated with poor CI performance (Wu, Lin, et al., 2015).
Understanding genetic causes of HL can determine the pattern and course of a patient's HL and may also predict outcomes after CI.The deficiency of this study is that the surgical outcomes of patients were not followed up, and we will make up for this weakness in the future.The mitochondrial 12S rRNA (ribosomal RNA) gene variant is associated with maternal inheritance, and the application of aminoglycosides resulted in irreversible hearing loss (Chen et al., 2013).The individuals who have 12S rRNA gene variant are sensitive to aminoglycoside drugs, and they can have normal hearing abilities by avoiding the use of aminoglycoside drugs.In the human mtDNA genome, m.1555A > G is the most common mutation in this gene.Hu et al. detected 44 variants in 3541 participants with 12S rRNA variants, and the detection rate of mtDNA 12S rRNA was 1.24% (Hu et al., 2021).We detected 12S rRNA m.1555A > G variant and m.1494C > T in six and one patients, respectively.For these patients, we conducted genetic counselling to avoid deafness induced by the use of aminoglycosides.Therefore, by screening mtDNA 12S rRNA gene, people at high risk of drug-induced deafness can benefit.
Another important significance of the genetic diagnosis for NSHL is to prevent genetic HL through pregnancy and prenatal genetic diagnosis and counselling.Specific DNA sequencing of the pathogenic or likely pathogenic variant sites was carried out in 23 foetuses.Seven foetuses carried homozygous or compound heterozygous variants associated with autosomal recessive hearing loss, and this diagnosis helped to directly prevent the birth of seven HL children successfully.To date, 124 genes have been reported to be associated with non-syndromic HL (http:// hered itary heari ngloss.org/ ).Fifty-one are autosomal dominant, 77 are autosomal recessive and 5 are X-linked.The genes have high clinical and genetic heterogeneity.Thus, the prenatal diagnosis should not only focus on identifying monogenic causes of disease, but also on the development of a detection strategy for digenic and oligogenic causes of disease which should be considered in the future.For example, ID/F type Usher syndrome (OMIM # 601067) is caused by double heterozygous variants of CDH23 and PCDH15.In our study, the patient's family would not give birth to patients with HL caused by LOXHD1 and MYO15A according to the rule of autosomal recessive inheritance.However, the couple had the heterozygous variants of CDH23 and PCDH15, respectively, and the foetus had both heterozygous CDH23 and PCDH15 variants simultaneously.This combination of double heterozygous variants has been reported (Zheng et al., 2005), and we avoided the birth of a child likely to have hearing loss.
In conclusion, we used the screening of variants in 15 hot spot mutations and WES for genetic diagnosis of 362 NSHL probands.We identified 20 novel variants in 15 deafness genes, which enlarged the variant spectrum of deafness genes in the Han Chinese population.In addition, our study helped to inform the genetic diagnosis and prenatal genetic diagnosis of deaf couple, and added to the theoretical basis for deafness diagnosis, accurate genetic counselling and the procreation guidance.
Abbreviations: −, not included in the gnomAD database; F, female; M, male; M, month; Y, years old.

Pathogenicity National Center for Biotechnology Information (NCBI)
Abbreviations: LP, likely pathogenic; P, pathogenic; VUS, variant of uncertain significance.
Pathogenicity analysis of novel variants.
Prenatal diagnosis analysis of the 23 carrier couples in this study.
T A B L E 4Abbreviation: N, no mutation.